Defined co-cultures of glutamatergic and GABAergic neurons with a mutation in DISC1 reveal aberrant phenotypes in GABAergic neurons

Background Mutations in the gene DISC1 are associated with increased risk for schizophrenia, bipolar disorder and major depression. The study of mutated DISC1 represents a well-known and comprehensively characterized approach to understand neuropsychiatric disease mechanisms. However, previous studies have mainly used animal models or rather heterogeneous populations of iPSC-derived neurons, generated by undirected differentiation, to study the effects of DISC1 disruption. Since major hypotheses to explain neurodevelopmental, psychiatric disorders rely on altered neuronal connectivity observed in patients, an ideal iPSC-based model requires accurate representation of the structure and complexity of neuronal circuitries. In this study, we made use of an isogenic cell line with a mutation in DISC1 to study neuronal synaptic phenotypes in a culture system comprising a defined ratio of NGN2 and ASCL1/DLX2 (AD2)-transduced neurons, enriched for glutamatergic and GABAergic neurons, respectively, to mimic properties of the cortical microcircuitry. Results In heterozygous DISC1 mutant neurons, we replicated the expected phenotypes including altered neural progenitor proliferation as well as neurite outgrowth, deregulated DISC1-associated signaling pathways, and reduced synaptic densities in cultures composed of glutamatergic neurons. Cultures comprising a defined ratio of NGN2 and AD2 neurons then revealed considerably increased GABAergic synapse densities, which have not been observed in any iPSC-derived model so far. Increased inhibitory synapse densities could be associated with an increased efficiency of GABAergic differentiation, which we observed in AD2-transduced cultures of mutant neurons. Additionally, we found increased neuronal activity in GABAergic neurons through calcium imaging while the activity pattern of glutamatergic neurons remained unchanged. Conclusions In conclusion, our results demonstrate phenotypic differences in a co-culture comprising a defined ratio of DISC1 mutant NGN2 and AD2 neurons, as compared to culture models comprising only one neuronal cell type. Altered synapse numbers and neuronal activity imply that DISC1 impacts the excitatory/inhibitory balance in NGN2/AD2 co-cultures, mainly through increased GABAergic input. Supplementary Information The online version contains supplementary material available at 10.1186/s12868-024-00858-z.


Background
The human cortex is primarily composed of excitatory and inhibitory neurons organized within the cortical local microcircuitry comprising inhibitory GABAergic interneurons to control excitatory, glutamatergic principal neuron output [1].GABAergic control may contribute to the synchronization of neuronal networks which is important for proper cognition, memory, and learning [2].Imbalance of excitation and inhibition (E/I imbalance) in the local microcircuitry is considered as one factor to contribute to a variety of neurodevelopmental disorders such as autism and schizophrenia.Therefore, the study of pathological mechanisms addressing E/I imbalance in neurodevelopmental disorders requires human models enabling interaction of excitatory and inhibitory neurons with each other.
With the possibility to generate induced pluripotent stem cells (iPSCs) and to differentiate these cells into specific neuronal phenotypes, an opportunity developed to create co-culture models comprising excitatory and inhibitory neurons harbouring a human genetic background.Different approaches have been proposed in the past to establish such models.One is based on differentiation with exogenously added factors providing a less defined network of excitatory and inhibitory neurons [3,4].Another strategy relies on directed differentiation of either excitatory or inhibitory neurons by endogenous expression of inducible transcription factors NGN2 or ASCL1/DLX2 (AD2), respectively [5,6].Co-culture models using the latter protocol enable the study of neuronal networks [7][8][9] after combination of different cell types at highly defined ratios that are more suitable to study E/I imbalance in neurodevelopmental disorders.Moreover, it has been reported that upon co-culture with NGN2 neurons, AD2 neurons show improved morphological and functional maturity [10].As an example, a recent study made use of such a defined network of induced glutamatergic and GABAergic neurons to study functional and morphological consequences of loss-offunction mutations in the SCZ risk gene SETD1A on single-cell and network level [11].
The DISC1 gene (Disrupted-in-schizophrenia 1) was originally identified in a large Scottish family with a balanced chromosomal t (1;11) translocation [12].Mutation of DISC1 predisposes to severe psychiatric disorders including schizophrenia, major depressive disorder, or bipolar disorder.The DISC1 protein is a scaffolding protein interacting with at least 16 other proteins [13] contributing to a variety of cellular or more specifically synaptic functions such as mRNA, synaptic vesicle and mitochondrial trafficking, as well as dendritic spine stabilization and synaptic plasticity.
To date, DISC1 mutations were used as a model for psychiatric disorders and E/I imbalance, mainly in rodent models.A critical contribution of GABAergic interneurons in rodent models is well established [14,15].On the other hand, human iPSC-derived neurons were applied to study DISC1 functions in human neurons after growth factor induction [3,[16][17][18][19].However, the crucial contribution of interneurons was not reported in any of these models.
Our study aims at the phenotypic evaluation of an iPSC-based co-culture system comprising NGN2-transduced cultures, enriched for glutamatergic neurons, cocultured with ASCL1/DLX2 (AD2)-transduced cultures, enriched for GABAergic neurons, as a model for excitatory-inhibitory neuron interaction.An isogenic pair of iPSC lines, one with a heterozygous DISC1 mutation in exon 2 (DISC1 ±), were comprehensively characterized to ensure that the DISC1 mutation line shows representative phenotypical features of DISC1 deficiency, which have previously been described.When cultured separately, excitatory synapses were reduced in NGN2 neurons, whereas inhibitory synapses in AD2 neurons remained unaffected.In a defined co-culture system of NGN2 and AD2 neurons, however, the E/I balance was shifted due to an increase of inhibitory synapses.In parallel, the activity of AD2 neurons was increased.

Methods iPSC line generation and maintenance
As described in Heider et al. (2021), the DISC1 ± iPSC line (TMOi001-A-5) was generated by the introduction of a heterozygous frameshift mutation in exon 2 by CRISPR-Cas9 gene-editing of a healthy iPSC line ( TMOi001-A).The isogenic parental line was obtained from Thermo Fisher Scientific (Gibco ™ episomal hiPSC line #A18945).iPSC lines used in this study are fully characterized, chromosomally intact and express stem cell markers on DNA and RNA level.iPSC were maintained in mTeSR Plus medium (STEMCELL Technologies, #100-0276) on Matrigel (Corning, # 354277)-coated 6-well plates.The cells were enzymatically passaged for single-cell seeding using Accutase (Sigma-Aldrich, # A6964).

NPC differentiation and maintenance
iPSC were differentiated into neural progenitor cells using the STEMdiff ™ SMADi Neural Induction Kit, following the embryoid body (EB) protocol (STEMCELL Technologies #08581).Briefly, iPSC were seeded at a density of 2 × 10^6 per well into AggreWell ™ 800 plates in STEMdiff ™ SMADi Neural Induction medium, pretreated with Anti-Adherence Rinsing Solution (STEM-CELL Technologies #07010).Daily medium changes were performed for seven days to allow the formation of EBs.Afterwards, EBs were harvested using 37 µm reversible strainers (STEMCELL Technologies #27215) and plated onto poly-l-ornithine/Laminin-coated 6-well plates.For coating, plates were treated with 20% poly-l-ornithine (PLO, Sigma-Aldrich #P4957) in DPBS for 2 h at RT, then washed with DPBS and DMEM/F12, followed by incubation with Laminin (10 µg/ml, Sigma-Aldrich #L2020) in DMEM/F12 overnight at 37 °C.EBs were cultivated for seven days with daily medium changes in STEMdiff ™ SMADi Neural Induction medium.Afterwards, neural rosettes were selected with STEMdiff ™ Neural Rosette Selection Reagent (STEMCELL Technologies #05832) for 1.5 h at 37 °C and plated on PLO/Laminin-coated 6-well plates in STEMdiff ™ Neural Progenitor medium (STEM-CELL Technologies #05833).NPC were maintained in Neural progenitor medium for cultivation and enzymatically passaged using Accutase.NPC were used for neuronal differentiations until passage 10.

NPC proliferation and cell death
NPC were seeded at a density of 3 × 10 4 /cm 2 on Matrigel-coated 96-well plates in Neural Progenitor medium.After cell attachment, NPC were stained with 5 µM of SYTOX ™ Orange Nucleic Acid Stain (Thermo Fisher Scientific #S11368) for 30 min at 37 °C to label cells with compromised membranes.Afterwards, cells were washed DMEM/F12 and medium was changed back to Neural Progenitor medium.Cells were imaged in brightfield mode and dead cells in fluorescence mode for 3 days every 4 h using IncuCyte ® .NPC proliferation and cell death were analyzed using the IncuCyte ® Basic Analyzer.Confluence was normalized to t = 0 at every time point and cell death numbers were normalized to the respective confluence at every time point.Data were analyzed from 3 independent experiments with at least 5 wells imaged per replicate.

Immunocytochemistry
Cells were fixed with 4% PFA in PBS for 15 min at RT and washed 3 × with PBS.For blocking and permeabilization, cells were incubated with blocking solution (1 × blocking reagent BMB (Roche #11,112,589,001) + 0.1% Triton X-100 (Carl Roth #3051) in PBS) for 30 min at RT.Primary antibodies were diluted in blocking solution and incubated overnight at 4 °C.Next, cells were washed 3 × with PBS and secondary antibodies, diluted in blocking solution, were applied for 2 h at RT. Cells were again washed 3 × with PBS and stored in PBS.

Imaging and analysis of DISC1 protein expression
Imaging of DISC1 protein expression in iPSC and NPC was performed using the spinning disk confocal microscope Cell Observer SD (Zeiss, Oberkochen, Germany) with a plan-apochromatic 20 × objective.From 3 independent experiments, 3-5 fields of view (containing multiple cells) were imaged per well. 2 wells were imaged for each condition in every biological replicate.From each microscopic field, DISC1 mean fluorescence intensity of 4 individual cells was measured by hand-drawn regions of interest (ROI) using the software ZEN 3.0 (Zeiss).Background intensities were measured per microscopic field and subtracted from DISC1 intensity values, which were afterwards normalized to WT mean within individual biological replicates.Data points in the graphs represent the average fluorescence intensity of multiple cells measured from a single microscopic field.

Quantification of GABA-positive neurons
DIV28 NGN2 and AD2 neurons were stained against MAP2 and GABA and z-stack confocal images were acquired with the spinning disk confocal microscope, using a 40 × objective (Zeiss, Oberkochen Germany).Images were acquired from two wells per clone per differentiation and from two independent neuronal differentiations.5 images were acquired per well.Orthogonal maximum intensity projections of the images were created using the software Zen 3.0 (Zeiss).Using Fiji (Image J), up to 10 MAP2-positive somata per image were randomly selected from images showing only the MAP2 channel, with the multi point tool.The selections were saved as ROIs and loaded onto the image containing only the channel with GABA staining to quantify how many of the pre-selected somata were GABA-positive.Percentages of GABA-positive neurons were calculated per image.

Imaging and analysis of synaptic marker expression
Imaging of synaptic marker expression in DIV28 neurons was performed using the spinning disk confocal microscope Cell Observer SD (Zeiss, Oberkochen, Germany) with a plan-apochromatic 63 × oil immersion objective.Images were acquired from 3 to 5 independent neuronal differentiations. 2 wells were imaged for each condition in each replicate.Within each well, 10 fields of view (z-stack images) with comparable MAP2-network density were acquired.
Analysis of synaptic densities was performed using IMARIS Bitplane 10.0.0 (Oxford Instruments, Abigdon, UK) as show in Additional file 1: Figure S1.For every individual microscopic field, a 3D mask of the MAP2 signal was created used to filter all synaptic signals lying within the MAP2 volume.Synapse numbers within the MAP2 volume were then quantified using the spot detection tool.Intensity thresholds for spot detection and spot size limits were kept constant within individual biological replicates for each synaptic marker.To analyze the co-localization of pre-and postsynaptic markers, the IMARIS XTension plugin 'Spots Colocalize' was used.A maximum distance of 0.2 µm between pre-and postsynaptic spots was defined, corresponding to the resolution limit of the Cell Observer SD microscope.
To distinguish between synapses on either NGN2 or AD2 neurons, a 3D mask of the GFP signal of NGN2 neurons was created.Synaptic channels were again filtered to exclude spots localized outside the GFP mask.Quantification of synapse densities and co-localization analysis was performed as described above.Synapse densities on AD2 neurons were derived by subtraction of synapse numbers on NGN2 neurons from the total synapse number on the MAP2 network.To account for differences in neuronal network density, all synapse numbers were normalized to the volume of either the MAP2 mask (all neurons), GFP mask (NGN2 neurons), or the difference of both (AD2 neurons).
Synapse densities from individual fields of view within a single well were averaged and normalized to the WT synaptic marker densities.

Calcium imaging
DIV28 neurons were stained with 2 µM of Calbryte ™ 590 AM (AAT Bioquest #20,700) diluted in neuronal medium for 30 min at 37 °C, 5% CO 2 .Afterwards, cells were washed with DMEM/F12 and medium was replaced by neuronal medium.Before imaging, the neurons were incubated for 15 min at 37 °C, allowing them to recover.To record spontaneous single-cell calcium activity, neurons were imaged at 37 °C, 5% CO 2 using the spinning disc confocal microscope Cell Observer SD. 3 min recordings of multiple cells within a microscopic field were obtained from 4 independent neuronal differentiations.For each differentiation, 4 wells with 2 videos per well were recorded.For each microscopic field, a snapshot of the Calbryte ™ signal (staining all neurons) and GFP signal of NGN2 neurons was obtained to allow for cell-type specific analysis.Somatic calcium signals were measured from individual neurons by hand-drawing ROI with Fiji and extracting the mean fluorescence intensity over time.A minimum of 3 spontaneously active neurons (min.one calcium peak per recording period) of both subtypes (NGN2, AD2) were analyzed per recording.Extracted traces were further analyzed with the Origin 2015G Peak Analyzer module (OriginLab Corporation).Traces were normalized to baseline, peak detection threshold and minimum peak distance were kept constant across all replicates.Peak parameters (AUC, FHWM, Amplitude) were averaged across all peaks of a single neuron.Data points in the graphs are normalized to the WT mean and represent the mean of multiple cells per well.

Lysis and protein quantification
For DigiWest cultures without astrocytes were used.40 µl of RIPA lysis buffer (Thermo Fisher Scientific #89900) supplemented with cOmplete ™ mini protease inhibitor (Roche # 11836153,001) was added to cell pellets on ice.Cells were lysed for 30 min at 4 °C.Protein quantification was performed using in-gel staining.1 µl of each original cell lysate was diluted 1:10 in lysis buffer.10 µl of the respective aliquots were loaded onto a NuPAGE 4-12% Bis-Tris precast gel (Thermo Fisher Scientific # NP0336BOX) and run according to the manufacturer´s instructions.The gel was washed with water and proteins were stained with BlueBandit (VWR # K217-1L) for 1 h.The gel was de-stained over night with ddH 2 O before detection at a LI-COR (LI-COR, Bad Homburg, Germany) instrument.Analysis and protein quantification was performed using ImageStudio (LI-COR).

DigiWest protein profiling
DigiWest was performed as published [20].A graphical representation of the workflow can be found in Additional file 1: Figure S2.In brief, 12 μg of cellular protein was loaded on an SDS-polyacrylamide gel and sizeseparated using the commercial NuPAGE system (Life Technologies).Size-separated proteins were blotted onto a PVDF membrane and biotinylated on the membrane using NHS-PEG12-Biotin (50 µM) in PBST for 1 h.After washing with PBST and drying of the membrane, the individual samples lanes were cut into 96 strips of 0.5 mm width using an automated cutting plotter (Silhouette America, West Orem, UT, USA) each corresponding to a defined molecular mass fraction.Each of the strips was placed in one well of a 96-well plate and 10 µl elution buffer (8 M urea, 1% Triton-X100 in 100 mM Tris-HCl pH 9.5) was added.The eluted proteins were diluted with 90 μl of dilution buffer (5% BSA in PBS, 0.02% sodium azide, 0.05% Tween-20) and each of the protein fractions was incubated with 1 distinct magnetic colorcoded bead population (Luminex, Austin, USA) coated with neutravidin.The biotinylated proteins bind to the neutravidin beads such that each bead color represents proteins of one specific molecular mass fraction.All 96 protein loaded bead populations were mixed resulting in reconstitution of the original lane.Aliquots of the Digi-West bead-mixes (about 1/200th per well) were added to 96 well plates containing 50 µl assay buffer (Blocking Reagent for ELISA (Roche #11112589,001) supplemented with 0.2% milk powder, 0.05% Tween-20 and 0.02% sodium azide).Beads were briefly incubated in assay buffer and buffer was discarded.Primary antibodies (complete list can be found in Additional file 2: Table S1) were diluted in assay buffer and 30 μl were added per well.After overnight incubation at 15 °C, the bead-mixes were washed twice with PBST and species-specific PElabelled (Phycoerythrin) secondary antibodies (Dianova, Hamburg, Germany) were added and incubated for 1 h at 23 °C.Beads were washed twice with PBST prior to readout on a Luminex FlexMAP 3D.

Data analysis
For quantification of the antibody-specific signals, an Excel-based analysis tool was employed [20] that automatically identifies peaks of appropriate molecular mass and calculates the peak area (reported as accumulated fluorescence intensity = AFI).Signal intensity was normalized to the total amount of protein loaded onto one lane.The software package MEV 4.9.0 was used for heatmap generation [21] along with GraphPad Prism (Version 9.0.0).Wilcoxon tests were performed to compare protein expression in WT and DISC1 ± neurons, and proteins with significantly different expression levels (p < 0.05) were plotted in the heatmap.

Phenotypes of NPC with a heterozygous DISC1 mutation
For the analysis of a neuronal co-culture we chose DISC1 mutation as a reliable and extensively evaluated model for psychiatric disorders [16,17,19,[22][23][24].To this end, we generated isogenic iPSC after introducing a heterozygous mutation into exon 2 of DISC1.Cells were fully characterized, and off-target mutations excluded, as described previously [25].Theoretically, the frame shift mutation allows for the translation of a truncated version of full length DISC1 encompassing the first 88 amino acid residues before translation becomes terminated at a STOP codon after additional 39 irregular amino acid residues [25,26].The parental wild-type line and the mutated heterozygous line (DISC1 ±) were differentiated into neuronal progenitor cells (NPC) and submitted to immunocytochemistry analysis to evaluate DISC1 expression using an antibody specific for the COOH terminus of DISC1 (Fig. 1A).Quantification of the fluorescence signals revealed a partial reduction of DISC1 staining both in iPSC and in NPC in accordance with a heterozygous mutation (Fig. 1B, C).In addition, Western blotting using an antibody directed against the N-terminus of the DISC1 protein was performed in iPSC (Additional file 1: Figure S3).A band was detected at ∼ 95 kDa, corresponding to the L/Lv-isoform of the DISC1 protein (Additional file 1: Figure S3A).Quantification of the ∼ 95 kDa band showed a partial reduction of DISC1 protein level in the DISC1 ± clone (WT = 1 ± 0.16, DISC1 ± = 0.77 ± 0.13, Additional file 1: Figure S3B).
Next, we performed a set of experiments to replicate previous observations and to assure a representative phenotype in our DISC1 ± line.Since DISC1 was suggested to play a role in neuronal proliferation [27], we tested NPC proliferation over time.Proliferation was increased for the DISC1 ± clone while analysis of cell death showed no apparent differences between the isogenic clones (Additional file 1: Figure S4).NPC were exposed to growth factors to induce neurite extension to be quantified with an IncuCyte ® device for unbiased, fully automated neurite outgrowth quantification.In accordance with previous studies, a reduction of neurite length in DISC1 ± neurons was observed (Fig. 1D, [24,28]).

Synapse densities in DISC1 ± NGN2 and AD2 neurons cultured separately
For further assessment of known DISC1 phenotypes, lentiviral transduction and overexpression of NGN2 in NPC was applied to generate populations enriched for glutamatergic, excitatory neurons [6] (Fig. 2A).At DIV 28, NGN2 transduced NPC gave rise to MAP2-positive neurons expressing co-transduced eGFP (Fig. 2B) as well as glutamatergic markers vGLUT1 and PSD95, which were partially co-localizing, indicating the formation of structurally intact glutamatergic synapses (Fig. 2C, C' , Additional file1: figure S5A).The MAP2 signal was used as a mask to identify synapse marker densities on neuronal cells.Quantification of excitatory synapse densities, as defined by apposition of presynaptic marker vGLUT1 with postsynaptic marker PSD95, revealed a significant decrease of glutamatergic synapses in DISC1 ± NGN2 neurons (Fig. 2D).In contrast, quantification of vGLUT1 and PSD95 clusters, separately, did not show significant differences (Fig. 2E, F).Thus, glutamatergic synapses are mostly affected in DISC1 heterozygous NGN2 neurons in agreement with a previous report [3].
Interestingly, when directly comparing WT and DISC1 ± AD2 cultures, a significant increase of the percentage of GABA-positive neurons was observed in mutant cultures (Additonal file 1: Figure S6C), suggesting enhanced differentiation efficiency towards the GABAergic lineage.WT and DISC1 ± cells expressed partially co-localizing GABAergic markers vGAT and gephyrin, indicating the presence of inhibitory synapses (Fig. 3C, C' , Additional file 1: Figure S5B).Quantification of GABAergic marker densities revealed no difference on WT and DISC1 ± neurons neither after evaluation of vGAT and gephyrin spots in apposition indicative for GABAergic synapses (Fig. 3D), nor after quantification of pre-or postsynaptic markers, separately (Fig. 3E, F).In conclusion, separate cultures of NGN2 or AD2 neurons reveal reduced glutamatergic synapse densities while inhibitory synapses did not show an overt phenotype.

Disturbed signaling pathways in DISC1 ± neurons
A highly sensitive, Western-Blot-based high-throughput approach for targeted protein profiling (DigiWest ® ) was applied to verify deregulation of known DISC1associated signaling pathways in differentiated neurons.Chosen target proteins included synaptic or neuronal markers, and members of well-known signaling pathways, directly or indirectly modulated by DISC1.In accordance, components of the WNT signaling pathway were included which regulate GSK3β activity along with beta-catenin abundance [30].Likewise, growth factor signaling proteins attributable to the Akt/mTOR, MAPK, PLC-γ and Jak/STAT pathways were examined which are pivotal to the DISC1-dependent regulation of diverse cellular functions such as differentiation and proliferation [31,32].In total, expression of a set of 88 proteins and phospho-proteins was studied (Additional file 2: Table S1), of which 68 showed detectable signals.Accumulated fluorescence intensity values (AFI) and normalized values can be found in Additional file 3: Table S2 and Additional file 4: Table S3.Of the 68 analytes with sufficient signals, 8 (~ 11.7%) were significantly deregulated in NGN2 DISC1 ± neurons and 5 (~ 7.3%) in AD2 DISC1 ± neurons suggesting differential regulation of proteins in the two populations of neurons (Additional file 1: Figure S7).
Analytes for which a consistent trend was observed across three independent neuronal differentiations are shown in Fig. 4A, B. Here, deregulated proteins of both neuronal phenotypes fall into the three major groups comprising WNT signaling, growth factor signaling, and neuronal proteins while differences in the expression of single group members do persist.It is of note that β-catenin and deshevelled (DVL2), involved in WNT signaling became deregulated in both NGN2 and AD2 neurons.Likewise, for growth factor signaling, NGN2 and AD2 neurons share deregulated expression of phosphorylated signal transducer and activator of transcription 3 (STAT3-pY705).Notably, expression of cell cycle regulator Cyclin D2 was increased in both NGN2 and AD2 neurons (Additional file 3: Table S2, Additional file 4: Table S3).In contrast, differences in WNT signaling between WT and DISC1 ± neurons were observed in NGN2 neurons regarding total and phosphorylated glycogen synthase-kinase 3β (GSK3β) and casein kinase 1ε, while casein kinase 1α was more deregulated in AD2 neurons.In the case of growth factor signaling, NGN2 neurons showed deregulated expression of RAC-alpha serine/threonine-protein kinase 1 (AKT1), phosphoinositide-dependent protein kinase 1 (PDK1), PDK1-phophoS241, GSK3α, phosphorylated phospholipase Cγ1 (PLCγ1-pS1248), PAK1/2/3, and STAT3-phosphoS272 while in AD2 neurons expression of phosphorylated PAK1/2-pS144/141, phosphorylated integrin-linked protein kinase 1 (ILK1-pS343), involved in cytokine and growth factor signaling, and mitogen-activated protein kinase 1 (MAPK1) was altered.
Interestingly, postsynaptic scaffolding protein PSD95 found in glutamatergic synapses was downregulated in NGN2 neurons while the postsynaptic scaffolding protein gephyrin was downregulated in AD2 neurons.Intermediate filament component vimentin was downregulated in DISC1 ± cultures of both cell types.Vimentin is expressed by various cell types in the human central nervous system, including neural progenitor cells, immature neurons and astrocytes [33].Since no astrocytes were added to the cultures for proteomic analysis and vimentin is replaced by neurofilaments in mature neurons [34], the expression in our cultures can likely be attributed to NPC or less mature neurons.. Differences between NGN2 neurons and AD2 neurons were observed regarding the deregulated expression of calcium/calmodulin dependent protein kinase IV (CAMKIV) and synapsin 1, respectively.In summary, protein profiling reveals that previously identified DISC1-mediated signaling became affected in the DISC1 ± line.
Thus far, we have assured using a series of different approaches, that the DISC1 ± line displays known phenotypic characteristics and therefore may serve as a representative cell line to mimic DISC1 deficiency for a comparison of separate cultures of NGN2 and AD2transduced neurons with a co-culture of both.

Co-cultures of DISC1 ± NGN2 and ASCL1/DLX2 neurons reveal E/I imbalance
For modelling the neuronal microcircuitry comprising excitatory and inhibitory neurons in a more defined and potentially more mature culture system, the two populations of NGN2 and AD2 neurons were subsequently mixed at a ratio of 80:20, resembling the approximate ratio of glutamatergic to GABAergic neurons reported in the cortex [35][36][37].NGN2-transduced neurons co-expressed eGFP to allow for discrimination from eGFP-negative, AD2-transduced neurons.Pre-and postsynaptic marker expression in conjunction with eGFP-positive and eGFP-negative cells was chosen as an approximation to assign synaptic structures to either NGN2 or AD2 neurons (Figs.5A, A' , B, B' , 6A, A' , B,  B').Overview images from which the zoomed-in image sections in Figs. 5 and 6 were obtained can be found in Additional file 1: Figures S8 and S9.
Quantification of presynaptic marker vGLUT1 as well as of postsynaptic markers PSD95 and gephyrin on eGFP-positive NGN2 neurons did not show any overt differences between WT and DISC1 ± neurons (Fig. 5C, D, F).In contrast, presynaptic vGAT spot density on NGN2 neurons was strongly increased by a factor of about 2.4 (Fig. 5E).Subsequently, synaptic marker densities were quantified on eGFP-negative AD2 neurons.Marker densities for glutamatergic input, namely vGLUT1 and PSD95 were not significantly altered on DISC1 ± AD2 neurons (Fig. 6C, D).However, GABAergic presynaptic input (vGAT) was increased by a factor of ~ fourfold while postsynaptic gephyrin cluster densities were unchanged (Fig. 6E, F).Next, we examined apposition of pre-and postsynaptic markers indicative of morphologically intact excitatory (vGLUT1 + PSD95) or inhibitory (vGAT + gephyrin) synapses (Additional file 1: Figure S10, Fig. 7).Separate analyses of NGN2 and AD2 neurons in co-culture revealed non-significant trends for less excitatory synapses and increased inhibitory synapses on NGN2 neurons (Additional file 1: Figure S10A, D).On AD2 neurons we observed no alterations regarding excitatory synapses (Additional file 1: Figure S10B) as well as slightly increased inhibitory synapses on AD2 neurons (Additional file 1: Figure S10E).However, synapse quantification in the whole MAP2-positive network revealed a significant increase in GABAergic synapse densities and no change in overall excitatory synapse densities (Additional file 1: Figure S10C, F).These alterations of synapse numbers result in a shift of the overall synaptic balance in the MAP2 neuronal network, leading to an E/I imbalance via increased inhibition (Fig. 7).The observed synaptic E/I imbalance is in line with previous findings in a mouse model with reduced DISC1 expression [15].

Calcium imaging reveals increased activation of DISC1 ± AD2 neurons
In a further set of experiments, we performed calcium imaging to monitor spontaneous neuronal activity in eGFP-positive, NGN2 neurons in comparison to eGFPnegative AD2 neurons.NGN2/AD2 co-cultures were loaded with a red emitting calcium sensor (Calbryte ™ 590 AM) (Fig. 8A) and calcium signals were recorded for 3 min (Fig. 8B).Calcium signals were measured at the soma of individual neurons, excluding astrocytic calcium signals due to the distinct morphology of the cells.Analysis of calcium signals of parental WT compared to mutated DISC1 ± neurons showed no changes of calcium parameters in NGN2 neurons (Fig. 8C-F).AD2 neurons showed an increase of peak amplitudes (ΔF/F0), while peak frequency, full-width half-maximum (FWHM) and area under the curve (AUC) were unchanged (Fig. 8G-J).Thus, the more pronounced activity of AD2, mostly inhibitory neurons, as compared to NGN2, mostly excitatory neurons, suggests functional imbalance in DISC1 ± cultures.

Discussion
iPSC-derived neurons provide a valuable tool to study disease mechanisms of psychiatric disorders in a relevant human model system.In this paper we aimed at the evaluation of a co-culture model comprising a defined ratio of NGN2 and AD2 neurons.Using DISC1 heterozygous neurons as an example, the co-culture revealed an impact of DISC1 mutation on GABAergic neurons at the morphological level which cannot be observed with separate cultures comprising either NGN2 or AD2 neurons only.A specific contribution was further confirmed by the observed selective activation of calcium signaling in AD2 neurons.Although it might represent an important mechanism contributing to the pathology of psychiatric disorders [38][39][40], GABAergic synaptic alterations of DISC1 mutant neurons were so far only studied in mice but not in models with human iPSC-derived neurons [15].

COOH-terminus
The divergent results in the NGN2/AD2 co-culture model as compared to cultures with only one neuronal type suggest an interaction of the two populations of neurons, contributing to the reported GABAergic phenotypes in DISC1 ± cultures.We observed enhanced differentiation efficiency of DISC1 ± AD2-transduced neurons towards the GABAergic lineage, which could contribute to the increase of GABAergic synaptic markers in co-cultures.Since this phenotype was not detected in separate cultures of AD2 neurons, the interaction with primarily glutamatergic NGN2 neurons in the co-cultures during the maturation period could exacerbate this intrinsic phenotype of AD2 neurons, resulting in excess formation of presynaptic terminals.In rodent models, the possibility of such an interaction has been previously documented after overexpression of mutant DISC1 in excitatory cortical neurons that decreased the density of parvalbumin-positive interneurons demonstrating a mechanism of how excitatory neurons can modulate the differentiation of interneurons, indirectly [43].In a further report, decreased DISC1 expression in immature granule cells, the principal neurons of the dentate gyrus, was shown to elicit increased GABAergic synapse formation [15].In a third study, knockdown of DISC1 in interneurons accounted for increased inhibitory GAD65positive synaptic terminals onto pyramidal neurons [14].Hence, mutant DISC1, both in excitatory and inhibitory neurons, increases inhibitory input onto excitatory neurons.Additionally, in the two latter reports, increased frequency of either miniature excitatory postsynaptic currents in interneurons or increased GABAergic spontaneous synaptic currents in excitatory neurons were observed, respectively.Taken together, this agrees with our findings in our human co-culture model where we found increased inhibitory presynaptic input onto NGN2 neurons and increased calcium signaling of AD2 neurons as an indication for enhanced GABAergic drive.
One limitation of our study is that we cannot exclude the presence of other neuronal cell types in NGN2 and AD2-transduced cultures.Our results suggest that while GABAergic neurons are almost absent in NGN2 cultures, only ~ 50% of neurons in AD2 cultures are GABA positive.While previous studies report high efficiency of GABAergic conversion of iPSC (~ 90% GABA-positive neurons) [10], the percentage of resulting GABA-positive cells has not been reported for AD2 transduction of NPC, which we chose as a starting cell type.To this end, it is important to optimize the differentiation protocol or prolong the cultivation period to obtain a more homogeneous population of GABAergic neurons in future experiments.Nevertheless, co-cultivation of NGN2 and AD2 transduced neurons could improve the maturity of the resultant cultures as suggested by previous reports [7,10].

Conclusions
In the present study, we used a pair of isogenic iPSC lines, one with a heterozygous mutation in DISC1, to investigate phenotypes of mutant NGN2 and AD2-transduced neurons.Initial experiments with NGN2 neurons cultured separately confirmed known DISC1-associated phenotypes, namely reduced excitatory synapse numbers and reduced neurite outgrowth, while inhibitory synapses of AD2 neurons remained unaffected.Targeted proteomic analysis revealed shared deregulation of proteins involved in Wnt signaling, growth factor signaling, and of neuronal proteins in both types of neurons.In co-cultures of NGN2 and AD2 neurons, we observed a strong GABAergic phenotype, which has not been reported before in an iPSC model.An overall increase in inhibitory synapses evoked a shift in E/I balance, which was supported by the finding of increased neuronal activity specifically in AD2 neurons.Overall, the NGN2/AD2 neuron co-culture model can serve as a suitable system to study early morphological and functional phenotypes of neuropsychiatric diseases in human iPSC-derived neurons.Our findings of aberrant phenotypes in GABAergic neurons provide an additional perspective for deciphering the role of DISC1 in neurodevelopmental disorders.

Fig. 7
Fig. 7 Ratio of excitatory to synapses (E/I) in co-cultures of NGN2 and AD2 neurons.A Quantification of synaptic densities, as defined by co-localized vGLUT1/PSD95 or vGAT/gephyrin clusters, in the MAP2-positive network of co-cultured NGN2 and AD2 WT and DISC1 ± neurons.Details on data points and statistics shown in Fig. 7 can be found in Additional file 1: Figure S5C, F. Y-axis values represent synapse marker spot numbers normalized to the MAP2 volume.Error bars: s.e.m